A New Phosphatidylinositol 4,5-Bisphosphate-binding Site Located in the C2 Domain of Protein Kinase Cα

Abstract

In view of the interest shown in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) as a second messenger, we studied the activation of protein kinase C␣ by this phosphoinositide. By using two double mutants from two different sites located in the C2 domain of protein kinase C␣, we have determined and characterized the PtdIns(4,5)P 2 -binding site in the protein, which was found to be important for its activation. Thus, there are two distinct sites in the C2 domain: the first, the lysinerich cluster located in the ␤3-and ␤4-sheets and which activates the enzyme through direct binding of PtdIns(4,5)P 2 ; and the second, the already well described site formed by the Ca 2؉ -binding region, which also binds phosphatidylserine and a result of which the enzyme is activated. The results obtained in this work point to a sequential activation model, in which protein kinase C␣ needs Ca 2؉ before the PtdIns(4,5)P 2 -dependent activation of the enzyme can occur.Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) 1 plays a key role in phosphoinositide signaling and regulates a wide range of processes at many subcellular sites. It is primarily detected in the plasma membrane but is also found in secretory vesicles, lysosomes, in the endoplasmic reticulum, the Golgi, and in the nucleus (1-5). PtdIns(4,5)P 2 can either bind to intracellular proteins and directly modulate their subcellular localization and activity, or it can act as a precursor for the generation of different second messengers. For example, several families of phospholipase C enzymes are responsible for the hydrolysis of PtdIns(4,5)P 2 in cells, leading to the production of diacylglycerol and inositol 1,4,5-trisphosphate (4, 6), which may, in turn, lead to the activation of different proteins such as some PKC isotypes.Protein kinase C (PKC) composes a large family of serine/ threonine kinases, which is activated by many extracellular signals and plays a critical role in many signal-transducing pathways in the cell (7-9). Based on their enzymatic properties, the mammalian PKC isotypes have been grouped into smaller subfamilies. The first group, which includes the classical isoforms ␣, ␤I, ␤II, and ␥, can be distinguished from the other groups because its activity is regulated by diacylglycerol (DAG) and, cooperatively, by Ca 2ϩ and acidic phospholipids, particularly phosphatidylserine (PS). Members of the second group are the novel mammalian (␦, ⑀, , and ) and yeast PKCs that are not regulated by Ca 2ϩ . The third group comprises the atypical PKC isoforms, , , and , whose regulation has not been clearly established, although it is clear that they are not regulated by DAG or Ca 2ϩ (8, 10). In classical PKC isoenzymes, Ca 2ϩ -dependent binding to membranes shows a high specificity for 1,2-sn-phosphatidyl-Lserine (11)(12)(13)(14). Additionally, this group of isoenzymes is sensitive to other anionic phospholipids, including phosphatidic acid and polyphosphoinositides (15-16) and to a variety of amphipathic membrane compounds, such as arachidonic acid and fre...

The F-actin Cross-linking and Focal Adhesion Protein Filamin A Is a Ligand and in Vivo Substrate for Protein Kinase Cα

“…In its Ca 2ϩ -bound state, this protein fold mediates an electrostatic interaction with acidic phospholipids, phosphatidylserine and phosphatidylinositol 4,5-bisphosphate (55,56), and translocates the kinase to the plasma membrane (47). In addition, Ca 2ϩ binding induces conformational changes in the regulatory domain of the kinase (47), thereby affecting its binding properties toward protein ligands (57).…”

Lipid binding domains: more than simple lipid effectors

“…Also of functional consequence is a cationic patch in the concave face of the bsandwich, termed the cationic b-groove, which varies in size and electrostatics among C2 domains (13). Cationic b-grooves have been shown to bind ceramide-1-phosphate (36) as well as PIs including PI(4,5)P 2 (37). Thus, many C2 domains are able to coordinate multiple lipids in both a Ca 21 -dependent or independent manner.…”

Specific Translocation of Protein Kinase Cα to the Plasma Membrane Requires Both Ca2+and PIP2Recognition by Its C2 Domain

“…Earlier studies have suggested that lysines 197 and 199 in the ␤3 strand and lysines 209 and 211 in the ␤4 strand of PKC␣C2 are important in Ca 2ϩ -independent, PIP 2 -dependent binding to vesicles, PSand PIP 2 -dependent regulation of PKC␣ activity in vitro, and membrane residence (Ochoa et al, 2002;Corbalan-Garcia et al, 2003;Rodriguez-Alfaro et al, 2004;Marin-Vicente et al, 2005). The hypothesis that these lysine residues are important in PIP 2 recognition predicts that mutations at these positions would weaken membrane-targeting specificity in vivo and lipid-binding affinity in vitro.…”

“…Domain-containing the PKC␣ C2 Ca 2؉ -binding Loops and the ␤3-4 Hairpin A basic region formed by lysine residues in the ␤-hairpin formed by ␤ strands 3 and 4 has been postulated to coordinate PS or PIP 2 in the membrane (Ochoa et al, 2002;Corbalan-Garcia et al, 2003;Rodriguez-Alfaro et al, 2004;MarinVicente et al, 2005), and an electron paramagnetic resonance study has demonstrated that this ␤3-4 hairpin lies in close proximity to the membrane surface (Kohout et al, 2003). To investigate the contribution of the ␤3-4 hairpin to specific Figure 5.…”

“…Previous studies have concluded that the CBLs are primarily or fully responsible for specific targeting to the plasma membrane in preference to other intracellular membranes (Stahelin et al, 2003;Marin-Vicente et al, 2005). In addition to the CBLs, a second membrane-interacting region consisting of basic residues in the ␤-hairpin formed by ␤ strands 3 and 4 has been identified and shown to interact with PS or another membrane lipid, PIP 2 (Verdaguer et al, 1999;Ochoa et al, 2002;Corbalan-Garcia et al, 2003). Mutations at key positions in the CBLs or in the ␤3-4 hairpin interfere with plasma membrane interaction in vivo and reduce the affinity of the PKC␣ C2 domain for PS or PIP 2 in vitro (Medkova and Cho, 1998;Ochoa et al, 2002;Bolsover et al, 2003;Stahelin et al, 2003;Rodriguez-Alfaro et al, 2004;Marin-Vicente et al, 2005).…”